A method for alkylating atosiban
By adsorbing atospermia with mesoporous nanomaterial KIT-6 molecular sieve and MIL-101 (Gr) type MOF, and carrying out disulfide bond reduction alkylation reaction, the problem of disulfide bond reduction difficulty in mass spectrometry detection was solved, and more efficient structural identification was achieved.
Patent Information
- Application Number
- CN202310660567.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-06
- Publication Date
- 2025-05-09
- Estimated Expiration
- 2043-06-06
AI Technical Summary
Atosiben showed resistance to fracture in mass spectrometry, and disulfide bond reduction was difficult to achieve, resulting in difficulty in identifying the drug structure of the peptide.
Mesoporous nanomaterials, such as KIT-6 molecular sieve and MIL-101 (Gr) type MOF, are used as solid phase support, by adsorbing atosepan and performing a reducing alkylation reaction of disulfide bonds on its surface, shortening the reaction time and improving the bialkylation efficiency.
The reduction alkylation reaction time of atosiben is significantly shortened and the proportion of bialkylation products is increased, thus making the structural analysis of atosiben more accurate and efficient.
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Figure CN116874566B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of drug detection, and particularly relates to an atosiban alkylation method. Background Art
[0002] Peptide drugs are a class of compounds composed of amino acids connected by peptide bonds. Since the introduction of insulin in 1922, more than 80 peptide drugs have entered the market for the treatment of a variety of diseases, including diabetes, cancer, osteoporosis, multiple sclerosis, HIV infection and chronic pain. Compared with chemical drugs, peptide drugs have higher specificity for in vivo target recognition, and are also more efficient and safe, and have become a new direction for new drug development. However, due to the cumbersome synthesis steps and unstable physical and chemical properties of peptide drugs, it is very necessary to identify their structure and impurity structure. High-resolution mass spectrometry has the advantages of high resolution and high mass accuracy, and has become one of the most powerful tools for identifying the structure of peptide drugs. Disulfide bonds are one of the most important post-translational modifications of proteins. They are connected by cysteine between or within chains, which is crucial for stabilizing the higher-order structure of proteins and regulating the biological functions of proteins. Therefore, the structure of peptide drugs often needs to construct disulfide bonds and add cross-links to stabilize the folded conformation, so as to exert their in vivo activity. However, since peptides or proteins with disulfide bonds show resistance to fragmentation in mass spectrometry detection, disulfide bond reduction is still a necessary condition for mass spectrometry to perform structural analysis through low-energy collision-induced fragmentation (CID). Therefore, the first condition for identifying peptide drugs by mass spectrometry is to reductively alkylate their disulfide bonds.
[0003] Atosiban, an oxytocin molecule modified at positions 1, 2, 4 and 8, is a competitive antagonist of oxytocin and vasopressin. It is commonly used in the clinic to treat premature delivery and to preserve pregnancy due to its low side effects. Its structure is c[Mpr-D-Tyr(OEt)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH2, and it is a disulfide bond-type cyclic polypeptide formed by the condensation of 9 amino acids, in which the thiol group of 3-mercaptopropionic acid at position 1 forms a disulfide bond with the thiol group of cysteine at position 6, stabilizing the structure and exerting biological functions. In the inspection work, we carried out reduction alkylation treatment on the raw material samples of atosiban, and then detected the samples by high-resolution mass spectrometry. The results showed that the proportion of dialkylated atosiban was relatively low, and the low abundance of dialkylated fragments made the structural analysis of atosiban very difficult. In response to this problem, we consulted relevant literature and found that the disulfide bond in the structure of atosiban is formed by the thiol group between 3-mercaptopropionic acid at position 1 and cysteine at position 6, while the intra-chain and inter-chain disulfide bonds of peptide drugs or protein molecules are usually formed by the thiol groups of two cysteines. Therefore, when two thiols with different activities undergo reductive alkylation, different reductive alkylation efficiencies may occur. 3-mercaptopropionic acid at position 1 is difficult to form an alkylation product, which makes it difficult to form a dialkylated atosiban. Failure to be reduced or alkylated successfully will directly affect the detection efficiency of the mass spectrometer, thereby affecting the structure and impurity detection of atosiban.
[0004] The main feature of molecular sieves is the narrow and symmetrical mesopore size distribution. They have been widely used as carriers for heterogeneous catalysis, selective adsorption and controlled drug release. The so-called confinement effect in porous materials strongly affects diffusion, phase change, catalytic performance, etc. As a mesoporous material, molecular sieve KIT-6 has a uniform, interpenetrating, bicontinuous three-dimensional cylindrical pore network with a narrow primary pore size distribution of 7 to 8 nm. At present, it has been proven to be an effective carrier for catalytic reactions.
[0005] MIL-101(Gr) is a metal-organic framework (MOFs). MOFs have the advantages of high porosity, large specific surface area, regular pores, and the metal ions they contain also have certain catalytic activity. Studies have used UIO-66-NH2 for the digestion of earthworm protein. Summary of the invention
[0006] Further optimization of the reductive alkylation conditions of atosiban to achieve the best double alkylation efficiency is very beneficial for the structural identification of atosiban or a polypeptide drug with a structure similar to atosiban.
[0007] The present invention has found through research that in order to effectively shorten the reaction time of atosiban reduction alkylation, using mesoporous nanomaterials as solid phase is a good choice. The inventor believes that mesoporous materials can effectively adsorb reactants and accelerate the reaction process of atosiban reduction alkylation.
[0008] The present invention uses KIT-6 molecular sieve / MIL-101 (Gr) type MOF to adsorb atosiban, and then adds dithiothreitol and iodoacetamide to carry out a reduction alkylation reaction of the disulfide bond, thereby shortening the reaction time. In addition, the dialkylated atosiban accounts for a high proportion, and the high abundance of dialkylated fragments makes the structural analysis of atosiban more accurate.
[0009] The invention provides an atosiban alkylation method, and a mesoporous material is used in the sulfhydryl reduction alkylation reaction of atosiban.
[0010] Furthermore, the mesoporous material includes one or more of porous carbon materials, MOFs, mesoporous molecular sieves and porous organic polymer materials.
[0011] Furthermore, the molecular sieve is KIT-6 molecular sieve, MCM-41, SBA-15, MCM-22; and the MOFs material is MIL-101 (Gr).
[0012] The present invention provides an atosiban alkylation method, comprising the following steps:
[0013] S1: adding the atosiban solution into the mesoporous material and shaking it sufficiently to allow the atosiban to enter the pores of the mesoporous material;
[0014] S2: adding a thiol reducing agent to carry out a reduction reaction;
[0015] S3: placing the reduced atosiban at room temperature, adding an alkylating agent in a dark environment, and performing an alkylation reaction;
[0016] Furthermore, the above method also includes:
[0017] S4: After the alkylation reaction, an organic solvent aqueous solution is added to the reaction solution to promote the release of atosiban from the mesoporous material after the reduction alkylation reaction, and separate the reaction solution from the mesoporous material.
[0018] Furthermore, in the above method, the thiol reducing agent is DTT, TCEP or BME, and the alkylating agent is iodoacetamide, chloroacetamide or cyclophosphamide.
[0019] The present invention provides an atosiban alkylation method, the method comprising:
[0020] S1: Add the atosiban solution to the KIT-6 molecular sieve to make the concentration of the molecular sieve 10 mg / mL, and shake it thoroughly to allow the atosiban to enter the holes of the molecular sieve.
[0021] S2: Add DTT solution and carry out reduction reaction at a certain temperature;
[0022] S3: Place the reduced atosiban at room temperature, add the IAM solution in a dark environment, and perform the alkylation reaction in a dark environment for a certain period of time under shaking at room temperature;
[0023] S4: Add 50% acetonitrile-water solution to the solution after the reaction, vortex, so that the atosiban after reduction and alkylation is completely released from the molecular sieve, centrifuge to separate the molecular sieve and the solution, and dilute and separate the supernatant with 50% acetonitrile-water solution.
[0024] The present invention provides an atosiban alkylation method, the method comprising:
[0025] S1: Add the atosiban solution to the MOFs material MIL-101 (Gr) to make the concentration of the MOFs material 10 mg / mL, and shake it fully to allow the atosiban to enter the holes of the MOFs material.
[0026] S2: Add DTT solution and carry out reduction reaction at a certain temperature;
[0027] S3: Place the reduced atosiban at room temperature, add the IAM solution in a dark environment, and perform the alkylation reaction in a dark environment for a certain period of time under shaking at room temperature;
[0028] S4: Afterwards, 50% acetonitrile-water solution is added to the solution after the reaction, and the solution is vortexed to completely release the reduced alkylated atosiban from the molecular sieve. The molecular sieve is separated from the solution by centrifugation, and the supernatant is separated by dilution with 50% acetonitrile-water solution.
[0029] The present invention provides an atosiban alkylation method, the method comprising:
[0030] S1: Add the atosiban solution to the mesoporous material mixture composed of MOFs material MIL-101 (Gr) and KIT-6 molecular sieve to make the concentration of the mesoporous material 10 mg / mL, and shake it fully to allow atosiban to enter the cavities of the mesoporous material;
[0031] S2: Add DTT solution and carry out reduction reaction at a certain temperature;
[0032] S3: Place the reduced atosiban at room temperature, add the IAM solution in a dark environment, and perform the alkylation reaction in a dark environment for a certain period of time under shaking at room temperature;
[0033] S4: adding 50% acetonitrile-water solution to the solution after the reaction, vortexing to completely release the reduced alkylated atosiban from the molecular sieve, centrifuging to separate the molecular sieve from the solution, and diluting the separated supernatant with 50% acetonitrile-water solution;
[0034] Furthermore, the ratio of MIL-101 (Gr) to KIT-6 molecular sieve in the mesoporous material mixture is 1:1 m / m.
[0035] Furthermore, in the above-mentioned atosiban alkylation method, the concentration of the thiol reducing agent DTT is 8-15 mM, the reduction temperature is 50-60° C., and the reduction time is 5-30 min, preferably 5 min; the concentration of the alkylating agent iodoacetamide is 10-30 mM, and the alkylation time is 3-15 min, preferably 3 min.
[0036] The beneficial effects of the present invention are: 1) adding KIT-6 molecular sieve and / or MIL-101 (Gr) to the reduction alkylation reaction of atosiban can shorten the reaction time through confinement effect; 2) optimizing and determining the DTT concentration, reduction time and reduction temperature in the reduction reaction of atosiban; and the IAM concentration and alkylation time in the alkylation reaction. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 . Effect of reducing agent concentration (a), reduction time (b) and reduction temperature (c) on atosiban reduction alkylation without adding KIT-6;
[0038] Figure 2 . Effect of alkylation agent concentration (a) and alkylation time (b) on reductive alkylation of atosiban without adding KIT-6;
[0039] Figure 3 Effect of reduction time (a) and alkylation time (b) on atosiban reduction alkylation after adding KIT-6;
[0040] Figure 4 Effect of reduction time (a) and alkylation time (b) on atosiban reduction alkylation after adding MIL-101;
[0041] Figure 5 Effect of reduction time (a) and alkylation time (b) on atosiban reduction alkylation after addition of KIT-6 and MIL-101;
[0042] Figure 6 Total mass spectrometric ion chromatogram of atosiban reductive alkylation reaction before optimization (a) and after optimization of reductive alkylation conditions based on KIT-6 molecular sieve promotion (b);
[0043] Figure 7 (a) Total ion current of atosiban alkylated at positions 1 and 6 and its corresponding secondary mass spectrum;
[0044] Figure 7(b) Mass spectra of atosiban when not reduced (a), reduced (b), monoalkylated (c), and dialkylated (d). DETAILED DESCRIPTION
[0045] Example 1
[0046] Example 1 is a comparative experiment.
[0047] This patent investigates the optimal concentration of DTT in the reductive alkylation reaction of atosiban without KIT-6 molecular sieves, controls other parameters except DTT concentration to remain unchanged, and investigates the dialkylation efficiency of atosiban when the DTT concentration is 8mM, 10mM, 12mM and 15mM; Figure 1 As shown in a, when the concentration of DTT is 8 mM, there is still a lot of atosiban that has not been reduced, and the relative peak area of dialkylated atosiban is only 87.37%. When the concentration of DTT is 15 mM, almost all of atosiban is reduced, and the relative peak area of dialkylated atosiban is 99.57%.
[0048] This patent investigates the optimal temperature in the reduction alkylation reaction of atosiban without KIT-6 molecular sieve, controls other parameters except the reduction temperature to remain unchanged, and investigates the dialkylation efficiency of atosiban when the reduction temperature is 50°C, 55°C and 60°C; Figure 1 As shown in Figure c, the best reductive alkylation efficiency can be obtained when the reaction temperature is 55 °C.
[0049] This patent investigates the optimal reduction time in the reductive alkylation reaction of atosiban without KIT-6 molecular sieve, controls other parameters except the reduction time to remain unchanged, and investigates the dialkylation efficiency of atosiban when the reduction time is 15min, 30min, 45min and 60min; the results show that with the extension of the reduction time, the relative peak area of dialkylated atosiban gradually increases, until the reduction time is 45min, almost all of the atosiban exists in the dialkylated form, and the dialkylated atosiban accounts for 98.6% of the total peak area ( Figure 1 b).
[0050] This patent investigates the optimal concentration of IAM in the reductive alkylation reaction of atosiban without KIT-6 molecular sieves, controls other parameters except the IAM concentration to remain unchanged, and investigates the dialkylation efficiency of atosiban when the IAM concentration is 10mM, 15mM, 20mM, 25mM and 30mM; the results show that as the concentration of IAM increases, the reductive alkylation efficiency of atosiban also increases ( Figure 2a), which is consistent with previous reports. When the concentration of IAM reached 30 mM, almost all of atosiban was converted to a dialkylated state, with a relative peak area of 99.57%.
[0051] This patent investigates the optimal alkylation time in the reductive alkylation reaction of atosiban without KIT-6 molecular sieve, controls other parameters except the alkylation time to remain unchanged, and investigates the double alkylation efficiency of atosiban when the alkylation time is 15min, 20min, 25min and 30min. The results show that ( Figure 2 b) When the alkylation time was 15 min, some mono-alkylated and unalkylated atosiban still existed, and the relative peak area of di-alkylated atosiban was only 87.23%. However, when the time was extended to 20 min, the di-alkylation efficiency of atosiban was significantly improved, and the relative peak area reached 98.91%.
[0052] Example 2
[0053] In order to further shorten the reaction time, this patent adds KIT-6 molecular sieve and examines the reduction time and alkylation time.
[0054] The results show that the dialkylation efficiency of KIT-6 molecular sieve-promoted reductive alkylation was significantly improved after the optimization of the conditions (see Figure 3 ):
[0055] 1 The reduction reaction of atosiban was carried out for 5 min, 10 min, 20 min and 30 min. The results showed that when the reduction time was 5 min, the dialkylation product could reach 99.69% (see Figure 3 (a)).
[0056] 2 The alkylation reaction of atosiban was carried out for 3min, 5min, 10min and 15min. The results showed that
[0057] When the alkylation time is 3 min, the product of double alkylation can reach 94.39% (see Figure 3 (b)).
[0058] 3. The addition of KIT-6 molecular sieve can greatly shorten the alkylation time of atosiban (reduction time is shortened from 45min to 5min; alkylation time is shortened from 20min to 3min; total reduction alkylation time is shortened from 65min to 8min.)
[0059] Example 3
[0060] MIL-101(Gr) is a metal-organic framework material (MOFs) with advantages such as high porosity, large specific surface area, and regular pores. Therefore, MIL-101(Gr) is expected to promote the efficiency of the reduction alkylation reaction of atosiban. In order to further shorten the alkylation reaction time, this example investigates the reduction time and alkylation time of atosiban under the promotion of MIL-101(Gr). The results show that
[0061] 1) The reduction reaction of atosiban was carried out for 5 min, 10 min and 15 min. The results showed that the dialkylation product could reach 98.88% when the reduction was carried out for 5 min (see Figure 4 (a)).
[0062] 2) The alkylation reaction of atosiban was carried out for 3 min, 5 min, 10 min and 15 min. The results showed that when the alkylation time was 3 min, the dialkylation product could reach 99.19% (see Figure 4 (b)).
[0063] 3) The addition of MIL-101(Gr) can greatly shorten the alkylation time of atosiban (from 65 min to 8
[0064] min).
[0065] Example 4
[0066] Based on the previous experiments, KIT-6 molecular sieve and MIL-101 (Gr) can be used to promote the reductive alkylation reaction of atosiban and shorten the reaction time. Therefore, the effect of KIT-6 molecular sieve and MIL-101 (Gr) participating in the reductive alkylation reaction of atosiban (1:1 m / m) was further investigated. The results showed that KIT-6 molecular sieve and MIL-101 (Gr) jointly promoted the reductive alkylation, and the double alkylation efficiency was significantly improved after the conditions were optimized (see Figure 5 ).
[0067] 1 After adding KIT-6 molecular sieve and MIL-101 (Gr), atosiban was reduced for 5min and 10min. The results showed that the dialkylation product could reach 99.77% when the reduction was 5min.
[0068] (See Figure 5 (a)).
[0069] 2 After adding KIT-6 molecular sieve and MIL-101 (Gr), the alkylation reaction of atosiban was carried out for 3min, 4min and 5min. The results showed that when the alkylation time was 3min, the double alkylation product could reach 99.65% (see Figure 5(b)).
[0070] 3 After adding KIT-6 molecular sieve and MIL-101(Gr), the total time of reductive alkylation was shortened from 65min to 8min.
[0071] Example 5
[0072] Comparison of alkylation efficiency of atosiban before and after optimization of reductive alkylation conditions based on KIT-6 molecular sieve promotion:
[0073] In this example, the mass spectrometry full scan detection was performed on the atosiban reduction alkylation products before and after optimization. The results are as follows: Figure 6 As shown, Figure 6 The total ion current peaks at 5.92 min (6a) and 5.89 min (6b) were dialkylated atosiban with a molecular weight of 1110.5068; Figure 6 The total ion current peaks at 6.26min and 6.69min in a are monoalkylated atosiban with a molecular weight of 1053.4840; Figure 6 The total ion current peak at 7.07 min in a is atosiban with reduced disulfide bonds, with a molecular weight of 996.4630; Figure 6 The total ion current peak at 7.28min in a is unreduced atosiban, with a molecular weight of 994.4474. Before optimization, the yield of dialkylated atosiban was very low, accounting for only about 40%, while after optimization, it was almost all dialkylated atosiban, successfully solving the problem that the special disulfide bond of atosiban is difficult to reduce and alkylate.
[0074] Example 6
[0075] Determination of retention time of 1-monoalkylated and 6-monoalkylated atosiban:
[0076] In order to explore whether the difficulty of atosiban in forming alkylated products is related to the special disulfide bonds in the structure of atosiban, the mass spectrum peak of monoalkylated atosiban with a mass-to-charge ratio of 1053.48 was subjected to secondary fragmentation. The results show (Figure 7) that the alkylated product formed by 3-mercaptopropionic acid at position 1 (RT: 6.25min) is significantly less than the alkylated product formed by cysteine at position 6 (RT: 6.66min). This indicates that the activity of 3-mercaptopropionic acid at position 1 is different from that of cysteine at position 6, resulting in different ratios of product formation. The low activity of 3-mercaptopropionic acid at position 1 in the alkylation reaction may be the reason why atosiban is difficult to form dialkylated products.
Claims
1. A method for alkylating atosiban, characterized in that: The method comprises: S1: Add the atosiban solution to the KIT-6 molecular sieve to make the concentration of the molecular sieve 10 mg / mL, and shake it thoroughly to allow the atosiban to enter the holes of the molecular sieve; S2: Add DTT solution and carry out reduction reaction at a certain temperature; S3: Place the reduced atosiban at room temperature, add the IAM solution in a dark environment, and perform the alkylation reaction in a dark environment for a certain period of time under shaking at room temperature; S4: Add 50% acetonitrile-water solution to the solution after the reaction, vortex, so that the atosiban after reduction and alkylation is completely released from the molecular sieve, centrifuge to separate the molecular sieve and the solution, and dilute and separate the supernatant with 50% acetonitrile-water solution.
2. A method for alkylating atosiban, characterized in that: The method comprises: S1: Add the atosiban solution to the MOFs material MIL-101 (Gr) to make the concentration of the MOFs material 10 mg / mL, and shake it fully to allow the atosiban to enter the holes of the MOFs material; S2: Add DTT solution and carry out reduction reaction at a certain temperature; S3: Place the reduced atosiban at room temperature, add the IAM solution in a dark environment, and perform the alkylation reaction in a dark environment for a certain period of time under shaking at room temperature; S4: Afterwards, 50% acetonitrile-water solution is added to the solution after the reaction, and the solution is vortexed to completely release the reduced alkylated atosiban from the molecular sieve. The molecular sieve is separated from the solution by centrifugation, and the supernatant is separated by dilution with 50% acetonitrile-water solution.
3. A method for alkylating atosiban, characterized in that: The method comprises: S1: Add the atosiban solution to the mesoporous material mixture composed of MOFs material MIL-101 (Gr) and KIT-6 molecular sieve to make the concentration of the mesoporous material 10 mg / mL, and shake it fully to allow atosiban to enter the cavities of the mesoporous material; S2: Add DTT solution and carry out reduction reaction at a certain temperature; S3: Place the reduced atosiban at room temperature, add the IAM solution in a dark environment, and perform the alkylation reaction in a dark environment for a certain period of time under shaking at room temperature; S4: adding 50% acetonitrile-water solution to the solution after the reaction, vortexing to completely release the reduced alkylated atosiban from the molecular sieve, centrifuging to separate the molecular sieve from the solution, and diluting and separating the supernatant with 50% acetonitrile-water solution; the ratio of MIL-101 (Gr) to KIT-6 molecular sieve in the mesoporous material mixture is 1:1 m / m.
4. The method according to any one of claims 1 to 3, characterized in that: The concentration of the thiol reducing agent DTT is 8-15 mM, the reduction temperature is 50-60°C, and the reduction time is 5-30 min; the concentration of the alkylating agent iodoacetamide is 10-30 mM, and the alkylation time is 3-15 min.